Background/Objectives: The rapid rollout of 5G has renewed interest in potential reproductive effects of mid-band (3.5 GHz) and millimeter-wave (24 GHz) radiofrequency electromagnetic fields (RF-EMF). We examined frequency- and duration-dependent changes in testicular cytokines, apoptosis-related genes, and sperm quality in rats. Methods: Male Sprague Dawley rats (n = 6 per group) were exposed for 60 days to 3.5 GHz or 24 GHz RF-EMF for 1 h/day or 7 h/day. The sham controls were housed identically. Testicular expressions of IL-10, IL-6, IL-1β, and TNF-α were quantified; Tp53, Bax, Bcl2, and Casp3 mRNA expressions were measured; and sperm concentration, viability, and motility were evaluated. Results: IL-10 was significantly reduced in the 24 GHz group at both 1-h and 7-h exposure duration. At 7 h, TNF-α was also lower at 24 GHz. Casp3 expression was higher and Tp53 was lower at 3.5 GHz at 1-h exposure duration. Sperm concentration and viability were reduced after 24 GHz exposure at 7 h, while sperm motility was reduced after 3.5 GHz exposure at both durations. Conclusions: Exposure to RF-EMF 3.5 GHz primarily impacts sperm motility via extrinsic pro-apoptotic pathways, while exposure to 24 GHz impacts sperm concentration and viability potentially through immune–apoptotic mechanisms, with all negative effects amplified by 7-h daily exposure.

The expansion of fifth-generation (5G) wireless networks has increased environmental exposure to mid-band and millimeter-wave radiofrequency electromagnetic fields (RF-EMF), but their molecular effects on male reproductive tissues remain insufficiently understood. This study evaluated whether repeated exposure to 3.5 GHz and 24 GHz RF-EMF alters testicular stress-associated molecular responses by integrating electromagnetic dosimetry with an in vivo rat model. Whole-body specific absorption rate (SAR) and 10 g peak SAR were estimated using a rat voxel model and scaled to the 20 cm antenna-to-cage geometry used during exposure. Thirty-six adult male Sprague Dawley rats were allocated to sham, 3.5 GHz, or 24 GHz groups and exposed for 1 h/day or 7 h/day over 60 days. Testes were examined histologically and assessed for HSP27, HSP70, and HSP90 protein expression. SAR values were low overall, although absorption was higher at 3.5 GHz than at 24 GHz. Histological evaluation showed preserved seminiferous tubule architecture without consistent structural injury. In contrast, molecular analysis demonstrated frequency- and duration-dependent modulation of heat shock proteins, including early HSP70 downregulation at both frequencies, followed by HSP90 upregulation at 3.5 GHz and HSP27 upregulation at 24 GHz. These findings indicate that low-level 5G-relevant RF-EMF exposure can modify molecular stress responses in testicular tissue even in the absence of overt histological damage.

The brain and testes are connected via the hypothalamic–pituitary–gonadal (HPG) axis. Both are vulnerable to radiofrequency electromagnetic radiation (RF-EMR). However, no comprehensive study had evaluated the effects of RF-EMR on key hormones along this axis. Hereby, this study evaluated the effect of RF-EMR on the hormonal changes along the axis, including the neuropeptide kisspeptin. A total of 18 (N = 18) adult Sprague–Dawley rats were divided into three groups (n = 6): Control, 4 h, and 24 h. The Control group was sham-exposed to an inactive router. The exposed groups were subjected to 2.45 GHz RF-EMR for 4 and 24 h daily, for 60 days at a 20 cm distance. The power density was 0.141 W/m² with a whole-body specific absorption rate (SAR) of 0.41 W/kg. No significant changes were observed in hypothalamic Kiss1 gene expression or serum kisspeptin levels. GnRH levels increased significantly in both exposed groups, while FSH and LH remained unchanged. Testicular testosterone was significantly reduced in the 24 h group, while serum testosterone was elevated in the 24 h group compared to the 4 h group. In conclusion, prolonged 2.45 GHz RF-EMR exposure caused selective changes in components of the HPG axis, particularly involving GnRH and testosterone, suggesting potential endocrine effects on male reproductive regulation.